3/15/2013

Skrining bioaktivitas bahan alam

sebagai antibakteri
NLP Vidya Paramita, S. Farm., M.Sc., Apt

Jurusan Farmasi FMIPA Udayana

Pertemuan IV : 15 Maret 2013

Antibiotic Susceptibility Testing (AST)

The purpose of performing antimicrobial
susceptibility testing (AST) is to provide in vitro
data to help ensure that appropriate and adequate
antimicrobial therapy is used to optimize treatment
outcomes
the AST data generated daily can be statistically
analyzed on an annual basis to generate an
antibiogram that reflects the antimicrobial
susceptibility and resistance patterns of important
pathogens that prevail in a particular hospital.

antibiogram
An antibiogram is the result of a laboratory
testing for the sensitivity of an isolated bacterial
strain to different antibiotics. It is by definition
an in vitro-sensitivity.

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Continued

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Klebsiella sp.
Continued antibiogram

Continued

E. coli
antibiogram

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Disk diffusion
The principle behind the disk diffusion method, or
Kirby Bauer test, is the use of a paper disk with a
defined amount of antibiotic to generate a
dynamically changing gradient of antibiotic
concentrations in the agar in the vicinity of the disk
Paper disk (6 mm)
The disk is applied to the surface of an agar plate
inoculated with the test organism; and while the
antibiotic diffuses out of the disk to form the
gradient

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Continued
The similar procedure is carried out in E-test,
where stripes are used instead of discs
In the cylinder method, stainless steel or
porcelain cylinders of uniform size
(usually8mm6mm10mm) are placed on the
inoculated agar surface of a Petridish,and filled
with samples and standards. After
incubation,the cylinders are removed and the
inhibition zones are measured

Continued
In the hole-plate assay, a few millimeter
diameter holes are cut in the inoculated agar
surface and filled with the samples. The tested
compound solution diffuses into
agarmediumcausing growth inhi- bition of the
microorganisms. The Petri dishes are left at
room temperature, prior to incubation. Then,
the zones of growth inhi- bition are measured

Continued
diffusion methods are less suitable to determine
the MIC values than dilution ones, because it is
impossible to measure the amountof the
testcompounddiffused into the agarmedium

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Continued...
A density of approximately 108 CFU mL1
(CFU = colony-forming unit) of the test organism
in the inoculum suspension is used to obtain
semi-confluent growth on the agar. Nnn
The inoculum is prepared by suspending enough
well-isolated colonies from an 18- to 24-hr agar
plate in broth or physiologic saline (0.85%) to
achieve a turbidity matching a 0.5 McFarland
Standard

Continued...
Mc farland standart = are used as a reference to adjust
the turbidity of bacterial suspensions so that the number of bacteria
will be within a given range. Original McFarland standards were
mixing specified amounts of barium chloride and sulfuric
acid together

Continued...
The inoculated plate is then allowed to dry with
the lid left ajar for no more than 15 min. Once
the agar plate is completely dry, the different
antibiotic disks are applied either manually or
with a dis-pensing apparatus.
In general, no more than twelve disks should be
placed on a 150-mm agar plate or five disks on a
90-mm plate

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The inoculum suspension should be used within 15
min of preparation. This is particularly important
for fastidious organisms that lose their viability
rapidly
A sterile cotton swab is dipped into the suspension
and pressed firmly on the inside of the tube to
remove excess liquid.
The dried surface of the appropriate agar plate is
inoculated by streaking the entire surface and then
repeating this twice, rotating the plate 60 each
time. This will result in an even distribution of the
inoculum.

Continued

Agar disc(on the left) and agar cylinder(on the right) method.
Testbacteria: Bacillus subtilis.

Continued
Interpretasi hasil uji aktivitas antibakteri :
1. Zona radikal = zona jernih disekitar paper disk
menandakan adanya aktivitas mematikan dari
senyawa uji
Zona irradikal = zona pertumbuhan bakteri
yang tidak rapat, menandakan adanya aktivitas
hambatan dari senyawa uji

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Continued
Pengukuran zona hambatan = diukur diameter
hambatan dengan penggaris atau dengan
jangka sorong .
Nilai zona hambatan : a+b+c
b c 3

a * dikatakan memiliki daya hambat jika

nilainya > 6 mm

Dilution
The main advantage of dilution methods is possibility to esti- mate the
concentration of the test compound in the agar medium or in the broth
suspension; for this reason, they are commonly used for determination
of MIC values
In the agar dilution procedure, various
concentrations of the tested compound are
mixed with a nutrient agar. The agar plates are
inoculated and then incubated. The lowest
concen- tration of the antimicrobial substance,
at which no microorganism growth is detected,
gives the MIC value.

Continued
In the tube assay, various concentrations of the
tested compound are mixed with bacterial
suspension in series of tubes the lowest
concentration causing inhibition in
microorganism growth corresponds to the MIC
value.

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microdilution
In the broth micro-dilution assay, the
microorganisms are grown in the plate wells, to
which various concentrations of the tested com-
pound are added. The growth of the microorganisms
is indicated by the presence of turbidity in the wells

Jurnal.....
Antibacterialandantifungalactivities of
neocryptolepine,biscryptolepineandcryptoquindolin
e, alkaloidsisolated from Crypto/epissanguino/enta

Continued
MIC = nilai konsentrasi terendah yang mampu
menghambat pertumbuhan bakteri
MBC = minimun baktericidal inhibition

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The Use of Thin-Layer Chromatography

with Direct Bioautography for
Antimicrobial Analysis
The main benefit of bioautography is that it
provides information about antimicrobial
activities of substances separated from a
mixture.
Bioautography methods are usually divided into
three categories:
1. Agar diffusion or contact bioautography.
2. Immersion or agar-overlay bioautography.
3. Direct bioautography.

Contact bioautography
In contact bioautography antimicrobials diffuse from a TLC
plate or paper to an inoculated agar plate.
The chromatogram is placed face down onto the inoculated
agar layer and left for some minutes or hours to enable
diffusion.
Then the chromatogram is removed and the agar layer is
incubated.
The inhibition zones are observed on the agar surface in the
places where the spots of antimicrobials are stuck to the agar.
The method resembles a disk assay.
The disadvantages of contact bioautography were difficulties
in obtaining complete contact between the agar and the plate
and adherence of the adsorbent to the agar surface.

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Immersion bioautography
In immersion bioautography the chromatogram is covered with a
molten, seeded agar medium.
After solidification, incubation and staining (usually with
tetrazolium dye) the inhibition or growth bands are visualized.
Sometimes, before incubation, plates are left for several hours at low
temperature to enable diffusion.
Agar-overlay is a hybrid of contact and direct bioautography.
Antimicrobials are transferred from the TLC plate to the agar layer
as in the contact assay but during incubation and visualization the
agar layer stays onto the plate as in direct bioautography.
The main disadvantage of this method is lower sensitivity caused by
dilution of antibacterials in the agar layer compared with direct
bioautography. Agar overlay is advised especially when direct
bioautography is impossible to perform.20

Direct bioautography
The principle of this method is that a developed TLC plate is dipped
in a suspension of microor-ganisms growing in a proper broth and
then incubated in a humid atmosphere.
A silica surface of the TLC plate covered with the broth medium
becomes a source of nutrients and enables growth of the
microorganisms directly on it.
However, in the places where antimicrobial agents were spotted, the
inhibition zones of the microorganism growth are formed.
Visualization of these zones is usually carried ou
usingdehydrogenase activity-detecting reagents; the most common
are tetrazolium salts.
The dehydro-genase of living microorganisms converts tetrazolium
salt into intensely colored formazan.
As a result, cream-white spots appear against a purple background
on the TLC plate surface, pointing the presence of antibacterial
agents

Continued
Metode bioautografi digunakan untuk
mengetahui letak dan jenis senyawa yang
memiliki aktivitas antibakteri
Dalam teknik pengerjaan bioautografi, Plat KLT
harus disiapkan sebanyak 2, dikembangkan
dengan kondisi dan waktu yang sama. 1 plat
untuk digunakan dalam uji aktibakteri. 1 plat
lainnya sebagai pembanding untuk
mendapatkan informasi golongan senyawa yang
memiliki aktiv itas antibakteri.

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Continued
Hasil yang didapatkan pada uji bioautografi :
Senyawa uji yang memiliki aktivitas antibakteri
akan menunjukkan spot jernih diantara latar
belakang ungu (dgn reagen tetrazolium salt) atau
spot jernih diantara latar belakang keruh (contact
bioautography).
Di hitung hRf spot yang memiliki aktivitas
antibakteri. Kemudian dilakukan identifikasi
golongan senyawa atau isolasi lebih lanjut
berdasarkan pada plat 2 berdasarkan hasil hRf spot
pada plat 1.

Jurnal
Antibacterial activity of Thai edible plants
against gastrointestinal pathogenic bacteria and
isolation of a new broad spectrum antibacterial
polyisoprenylated benzophenone, chamuangone
An Antimicrobial Compound Isolated from
Cinnamomum Iners Leaves with Activity against
Methicillin-Resistant Staphylococcus Aureus

Kesimpulan
Pemilihan bakteri uji, ditentukan oleh tujuan
riset yg akan dilakukan :
Contoh :
1. uji aktivitas antibakteri penyebab diare :
bakteri gram negatif (E. coli, Salmonella sp.,
Shigella sp.)
2. Uji antiacne : bakteri penyebab acne
3. Uji antibakteri topikal : bakteri yang ada di
topikal

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Continued
Pemilihan media, waktu dan suhu inkubasi untuk
tumbuh tergantung bakteri yang digunakan. Pilih
media yang sesuai yang mampu menumbuhkan
bakteri secara optimum
Pemilihan metode uji. Ada 3 metode : difusi, dilusi,
dan bioautografi
Bisa gabungan antara 2 dari ketiga metode yang
ada. Tergantung tujuan riset
Penentuan MIC hanya bisa dilakukan dengan
metode dilusi. Dan dengan uji bioautografi kita
dapat mengetahui letak dan jenis senyawa yang
berperan dalam aktivitas antibakteri

Continued
Satu uji curah kerja :
1. Kontrol normal = sebagai kontrol normal
pertumbuhan bakteri, jika tanpa diberi perlakuan.
Diaplikasikan pada metode dilusi
2. Kontrol negatif = sebagai kontrol negatif, dimana
diharapkan menghasilkan hasil uji yang negatif.
Yaitu pembawa/pelarut ekstrak uji seperti DMSO.
Bisa juga berupa pelarut organik yang mampu
melarutkan ekstrak uji, namun hanya dpt
dipalikasikan pada metode Kirby bauer

Continued
3. Kontrol positif = sebagai kontrol positif, dimana
diharapkan menghasilkan hasil uji yang positif.
Dipilih antibiotik yang sesuai utk terapi terhadap
bakteri uji yg digunakan.
4. Variasi konsentrasi senyawa uji =
Dalam uji aktivitas antibakteri, digunakan loading
dose.
Loading dose = jumlah dosis yg digunakan dalam
uji perlakuan antibakteri
perhitungan loading dose :
I. Konsentrasi ekstrak 10 mg/mL
10 g/ L,
loading dose dalam 1 paper disk (10 L)
100 g

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